Splenectomy has opposite effects on the growth of primary compared with metastatic tumors in a murine colon cancer model

The spleen is a key source of circulating and tumor-infiltrating immune cells. However, the effect of splenectomy on tumor growth remains unclear. At 3 weeks after splenectomy, we subcutaneously injected LuM1 cells into BALB/c mice and evaluated the growth of primary tumors and lung metastases at 4 weeks after tumor inoculation. In addition, we examined the phenotypes of immune cells in peripheral blood by using flow cytometry and in tumor tissue by using multiplex immunohistochemistry. The growth of primary tumors was reduced in splenectomized mice compared with the sham-operated group. Conversely, splenectomized mice had more lung metastases. Splenectomized mice had fewer CD11b+cells, especially monocytic MDSCs (CD11b+Gr-1neg-lowLy6chigh), and NK cells (CD49b+CD335+). The proportion of NK cells was inversely correlated with the number of lung metastases. In splenectomized mice, the density of CD3+ and granzyme B+ CD8+ T cells was increased, with fewer M2-type macrophages in primary tumors, but NK cells were decreased markedly in lung. Splenectomy concurrently enhances T cell-mediated acquired immunity by reducing the number of monocytic MDSCs and suppresses innate immunity by decreasing the number of NK cells. Splenectomy has opposite effects on primary and metastatic lesions through differential regulation on these two immune systems.

The spleen is a key source of circulating and tumor-infiltrating immune cells.However, the effect of splenectomy on tumor growth remains unclear.At 3 weeks after splenectomy, we subcutaneously injected LuM1 cells into BALB/c mice and evaluated the growth of primary tumors and lung metastases at 4 weeks after tumor inoculation.In addition, we examined the phenotypes of immune cells in peripheral blood by using flow cytometry and in tumor tissue by using multiplex immunohistochemistry.The growth of primary tumors was reduced in splenectomized mice compared with the sham-operated group.Conversely, splenectomized mice had more lung metastases.Splenectomized mice had fewer CD11b + cells, especially monocytic MDSCs (CD11b + Gr-1 neg-low Ly6c high ), and NK cells (CD49b + CD335 + ).The proportion of NK cells was inversely correlated with the number of lung metastases.In splenectomized mice, the density of CD3 + and granzyme B + CD8 + T cells was increased, with fewer M2-type macrophages in primary tumors, but NK cells were decreased markedly in lung.Splenectomy concurrently enhances T cell-mediated acquired immunity by reducing the number of monocytic MDSCs and suppresses innate immunity by decreasing the number of NK cells.Splenectomy has opposite effects on primary and metastatic lesions through differential regulation on these two immune systems.
The spleen is the largest lymphoid organ, and its numerous immune cells play complex roles in immune surveillance and immune defense against infection and cancer 1 .Splenectomy is often performed during upper abdominal surgery for trauma or cancer treatment.Because of recent advances in understanding tumor immunology, the exact contribution of splenectomy to patient outcome is attracting interest.Epidemiologic studies indicate that splenectomy for trauma has little, if any, effect on the subsequent occurrence of cancers 2,3 .However, some clinical studies suggest that splenectomy negatively affects the survival of patients with gastric 4,5 , colonic 6 , or pancreatic 7 cancers.Yet, these findings might reflect confounding bias associated with the clinical indication for splenectomy, including locally advanced tumors and increased surgical stress.Therefore, whether splenectomy increases the risk of cancer growth or recurrence in human patients is unclear.
Murine studies of the effects of splenectomy on tumor development and progression have yielded highly conflicting results.In some studies, splenectomy slightly suppressed the growth of subcutaneously 8,9 or orthotopically 10 inoculated tumors.Other studies have reported nonsignificant differences [11][12][13][14] or even tumorgrowth-promoting effects 15,16 .In metastatic models, splenectomy has been reported to either enhance 8,11,[14][15][16][17][18][19] or inhibit 9,13,[20][21][22] the formation of lung or liver metastases.These discrepancies have been attributed to the diversity of experimental models used, including differencies in tumor cell type, and mouse strain, and in the methods for evaluating anti-tumor effects.In particular, the effects of splenectomy on tumor growth are highly dependent on the timing of splenectomy relative to tumor inoculation 9,20,23,24 .In the same experimental series, splenectomy has differently affected the growth of primary tumors and metastases 8,11,13,14,17 .
The spleen is a key reservoir and source of circulating and tumor-infiltrating immune cells 25,26 .In particular, the frequencies of immunosuppressive myeloid-derived suppressor cells (MDSCs), which disrupt both adaptive

Splenectomy suppressed the growth of primary (subcutaneous) tumors
Tumors were detected in all mice at about 10 days after subcutaneous injection of LuM1 colon adenocarcinoma cells.Weight trends did not show significant differences between each group (Fig. 1B).Splenectomy prior to tumor inoculation slightly but significantly suppressed the growth of primary (subcutaneous) tumors (Fig. 1C, D).In the splenectomy group, the median tumor volume after euthanasia at day 28 (1400 mm 3 ; range, 174-3063 mm 3 , n = 16 per group) was significantly less than that of the sham control group (2604 mm 3 , 44-3934 mm 3 , n = 15, P < 0.05).Tumor weight after euthanasia was reduced in the splenectomy group (1280 mg, 230-2050 mg; controls: 1950 mg, 30-3440 mg, P < 0.05) also.

Splenectomy exacerbated spontaneous lung metastases
By day 28, multiple metastases had developed in the lungs of both groups.However, the median number of macroscopic metastases visible at the lung surface was significantly higher in splenectomized mice (100; range, 7-246) than in control mice (32, 0-181, P < 0.01) (Fig. 2A, B).We then used microscopy to evaluate the metastatic lesions including microscopic tumors in a representative coronal tissue section from each mouse.Again, the median number of lung metastases was significantly higher in the splenectomy group (58.5, 23-134) than in controls (9, 0-42, P < 0.01) (Fig. 2C, D).

Splenectomy decreased the frequency of natural killer (NK) cells in the peripheral blood of LuM1 tumor-bearing mice
Using flow cytometry, we calculated the ratios of T cells and NK cells in CD45(+) cells from peripheral blood at day 28 by gating on lymphocytes (Fig. 3A).Neither the frequencies of CD3 + , CD4 + , and CD8 + T cells nor CD4 + / CD8 + ratios differed between splenectomized and control mice (Fig. 3B-E).However, the median frequency
Recent studies have suggested that monocytes can be classified into inflammatory classical (CCR2 high Ly6C + ) and patrolling nonclassical (CX3CR1 + Ly6C -) subtypes, with different dynamics and functions 34 .Patrolling monocytes differentiate from classical monocytes through Notch signaling primarily in bone marrow and spleen 35 and play critical roles in the elimination of circulating tumor cells 36 .We therefore examined the frequencies of inflammatory and patrolling monocytes in peripheral blood.Most of the M-MDSCs showed the inflammatory CCR2 high Ly6C + phenotype, and their frequency was significantly lower in splenectomized mice (3.38%, 1.90-7.87%)than in controls (7.59%, 5.04-12.00%,P < 0.01) (Fig. 4E).The frequency of CX3CR1 + Ly6C -patrolling monocytes was also lower in the splenectomy group, but the difference between groups did not reach statistical significance (Fig. 4F).When analyzing the absolute cell numbers, splenectomy and control group exhibited a similar trend in every subpopulation, with a slightly more pronounced difference (Supplementary Fig. 1).

The number of lung metastases correlated with the frequencies of NK cells and patrolling monocytes in peripheral blood
We then assessed the correlations between the number of lung metastases and various types of circulating immune cells in all mice.The number of lung metastases showed a strong inverse correlation with the frequency of CD49b + CD335 + NK cells (r = -0.8646,P < 0.01) and CD11b + Gr-1 -Ly6C -CX3CR1 + patrolling monocytes (r = -0.6405,P < 0.05) (Fig. 5A, B), whereas the frequencies of NK cells and patrolling monocytes showed strong positive correlation (r = 0.7477, P < 0.01) (Fig. 5C).

Discussion
The influence of splenectomy on tumor development and progression remains inconclusive.In previous murine studies, splenectomy had both suppressive 9,10,[20][21][22][23]29 and promoting 8,11,[14][15][16]18,19 effects on tumor growth, suggesting that the role of the spleen in tumor behavior is largely dependent on cancer cell type and stage. Inthe present study, we used a spontaneous lung metastasis model and found that splenectomy prior to tumor induction had opposite effects on the growth of primary compared with metastatic lesions: splenectomy slightly suppressed the growth of the subcutaneous primary tumors but exacerbated lung metastases.We observed the same effects of    splenectomy on primary tumor and peritoneal metastasis using a gastric cancer cell, YTN16P, and C57BL/6N mouse (Supplementary Fig. 7). Differential effcts of splenectomy on primary compared with metastatic sites has previously been reported for other cancer cell types, including melanoma 17 , hepatoma 11 , lung carcinoma 8 , and breast cancers 13,14 .Together, these combined results suggest that splenectomy differently modulates the immune microenvironment in primary tumors compared with the metastatic or premetastatic niche in the target organ.
Consistent with previous studies 9,13,29 , we found that the frequency of peripheral myeloid cells tended to be decreased and the frequency of Ly6C high CCR2 + M-MDSCs was reduced after splenectomy; these findings confirm the concept that spleen is a major source of circulating and tumor-infiltrating MDSCs.Moreover, we found that M2-type TAMs in primary tumor were reduced and CD4 + and CD8 + tumor-infiltrating T cells were increased in splenectomized mice, although the frequencies of total T cells and their subsets in peripheral blood did not differ between groups.Considering that a significant proportion of the CD8 + TILs exhibit positivity for granzyme B, and their density also exhibited an increase in splenectomized mice, it is reasonable to designate them as the principal activated effector cells responsible for tumor reduction.Previous studies have shown a positive correlation between the CD8 + TILs and clinical outcome in many cancers, including colon cancer [37][38][39] .In addition, our results are supported by the fact that many in vivo experiments have shown increased tumor volume and poor prognosis with the depletion of CD8 + T cell using anti-CD8 antibody [40][41][42][43] .
Circulating M-MDSCs infiltrate tumors via the CCL2/CCR2 axis and differentiate into M2-type TAMs 44 ; this cell population plays a pivotal role in assisting tumor growth by suppressing T cell functions and promoting angiogenesis 28,45 .Blockade of the CCL2/CCR2 axis decreased tumor-infiltrating inflammatory monocytes-macrophages but increased T cell infiltration in a murine pancreatic cancer model 46 .Considering these previous results with our current findings raises the possibility that the decreased number of M-MDSCs after splenectomy may enhance T cell infiltration and activation in the tumor microenvironment, thus inhibiting the growth of primary tumors.
In contrast to the effects on primary tumors, splenectomy markedly accelerated lung metastasis.Similar results have previously been reported in some studies 8,11,14,20 but not others 9,13,21 .Tumor metastasis is regulated by various factors including cytokines, chemokines as well as bone marrow derived cells.In particular, proinflammatory cytokines secreted from tumor cells and host cells are known to play key roles in the formation of pre-metastatic niche 47 .In the current study, we additionally found that the frequency of NK cells, which were positive for CD49b and CD335 (also known as NCR1 and NKp46), was greatly reduced in peripheral blood and in both metastatic lesions and metastasis-free lung tissue in splenectomized mice.The majority of the NK cells in lung were positive for granzyme B, suggesting activated NK cells.Moreover, the number of lung metastases was inversely correlated with the ratio of NK cells.Given that NK cells negatively control metastasis through an NCR1-dependent mechanism [48][49][50] , the reduced number of NK cells after splenectomy likely is responsible for the metastasis-promoting effect we observed.Indeed, splenectomy reduces the number of NK cells and increased metastasis formation in the liver; spleen was proposed as a source of factors that allow the traffic of NK cells into metastatic lesions 18 .In contrast to our findings, splenectomy reduced lung metastases in other studies 9,13,21 .Although NK cells are well known to play a crucial role in controlling tumor metastasis, the sensitivity to NK cell cytotoxicity differs markedly depending on tumor cell types and their receptors they possess that control the function of NK cells [51][52][53] .In this study, the sensitivity of LuM1 cells to NK cells may have influenced the exacerbation of lung metastases due to the decrease in NK cells by splenectomy.In addition, metastases that arise from clusters of circulating tumor cells (CTCs) are much less sensitive to NK-mediated clearance than those that arise from single CTCs 54 .Moreover, NK cells effectively limit the pulmonary seeding of B16 melanoma cells, whereas T cells play a primary role in restricting the growth of metastatic foci in the lung 55 .Therefore, the apparently inconsistent effects of splenectomy on lung metastases may reflect the different sensitivities of various tumor cell types to NK cell-mediated lysis.The density of CD3 + CD8 + T cells in metastatic lung tumors was increased in splenectomized mice, showing the same trend as that in primary tumors, and likely was due to the reduced number of M-MDSCs.Therefore, had we followed the splenectomized mice longer, we might have observed decreased growth of their metastatic lesions due to the cytotoxic effects of CD8 + TILs.
The mechanisms leading to the reduction in the number of circulating NK cells after splenectomy are unclear yet.According to a recent report, spleen-resident immature NK cells undergo extramedullary maturation in a T-bet-dependent manner, and the spleen is a key source of NK cells that can circulate peripherally and then migrate into the tumor tissue 56 .Importantly, patrolling monocytes promote NK cell recruitment and activation, which leads to the elimination of tumor cells in the lung 36 .In our current study, the proportion of patrolling monocytes correlated positively with that of NK cells and inversely with the number of lung metastases.The number of patrolling monocytes tended to be reduced in splenectomized mice, although the difference did not reach statistical significance.Given that the spleen is considered to be one of the main sites for the differentiation of patrolling monocytes 35 , decreased NK activity may be related to a reduced number of patrolling monocytes.
In the present study, the effect of the timing of splenectomy on tumor development could not be investigated.Previous studies have reported that the timing of splenectomy influences tumor progression 9,20,23,24 .Soda et al. investigated the effect of the timing of splenectomy on the survival in a murine colon cancer model by performing splenectomy either before or after tumor inoculation.In their study, splenectomy significantly prolonged the survival when it was performed prior to tumor inoculation, but not when performed after inoculation 12 .We performed splenectomy 3 weeks before tumor inoculation to minimize the effect of surgical stress on tumor development, however, the results may have been different if surgery had been performed after inoculation.Moreover, the method of tumor inoculation would also affect the tumor progression.Most of these previous studies used metastatic mouse models established by intravenous or intrasplenic injection of cancer cells 8,19,21 .In the present study, we subcutaneously inoculated mice with LuM1 cells to create a spontaneous lung metastasis model, allowing the effect of splenectomy on the primary tumor and lung metastases to be evaluated together.Given the differences in the microenvironment between the subcutaneous and the orthotopic sites, as well as metastatic sites, orthotopic inoculation (i.e., colon) or intravenous injection of LuM1 cells may have led to different tumor development.Our result shows that further research is needed to understand the potential role of splenectomy in therapy.
In summary, we found that the growth of LuM1 subcutaneous primary tumors was suppressed slightly, with reduced numbers of systemic M-MDSCs and M2-type TAMs and increased numbers of TILs, after splenectomy in BALB/C mice.These findings suggest that splenectomy enhances T cell-mediated immunity by inhibiting the expansion and systemic supply of M-MDSCs.In contrast, splenectomy enhanced lung metastasis, with decreased numbers of NK cells in peripheral blood and lung tissue, indicating that splenectomy suppresses NK cell-mediated immunity.The overall influence of splenectomy on a tumor-bearing host depends on the balance between these two different immune responses.A precise understanding of the susceptibility of each tumor type to NK-or T cell-mediated immunity will enable the development of next-generation cancer immunotherapy strategies.

Cell culture
LuM1, a highly lung metastatic subclone of the murine colon adenocarcinoma 26 line, was a kind gift from Dr. K. Oguri (Aichi Cancer Center, Nagoya, Japan) 33 and was maintained in RPMI supplemented with 10% fetal bovine serum, 100U/mL penicillin, and 100 µg/mL streptolysin (Sigma-Aldrich, St. Louis, MO, USA).YTN16, a murine gastric cancer cell, established in p53 heterozygous knockout C57BL/6 mice by oral intake of N-Methyl-N-nitrosourea (MNU) was a generous gift from Dr S.Nomura (Tokyo University, Japan) 57 .YTN16P, a highly metastatic subline of YTN16 obtained through in vivo selection methods 58

Animal model
The experimental protocol is shown in Fig. 1A.Female BALB/c and C57BL/6N mice (age, 5 weeks) were purchased from CLEA Japan (Tokyo, Japan), housed under specific pathogen-free conditions, and underwent splenectomy (or sham operation) under anesthesia with isoflurane (Fujifilm Wako Chemical, Osaka, Japan) at 6 weeks of age as previously described 59 .Mice in the splenectomy group received an approximately 1-cm incision in the left flank, gentle mobilization of the spleen, ligature of splenic vessels, and removal of the spleen.In mice in the sham control group, the abdominal wall was opened, and the spleen was mobilized and manipulated gently for 3 min.Laparotomy wounds were closed in a continuous two-layer pattern.At 3 weeks after surgery, 1 × 10 6 LuM1 or YTN16P cells in 100 µL HBSS were injected subcutaneously in the right flank of each mouse.
Twice weekly thereafter, we weighed the mice, used calipers to measure the diameter of subcutaneous (primary) tumors, and calculated tumor volume (mm 3 ) as [tumor length (mm) × tumor width (mm) 2 ]/2.At 4 (LuM1) or 8 (YTN16P) weeks after tumor inoculation, mice were euthanized through deep anesthesia by isoflurane.Immediately after euthanasia, blood samples (800 µL) were collected from the inferior vena cava, and 2 investigators blinded to treatment group independently weighed the primary tumor and counted the number of macroscopic metastatic lung nodules; the average of the two values was adopted for each parameter.
As syngeneic peritoneal metastasis model, YTN16P (1 × 10 6 cells) was intraperitoneally injected into C57BL/6N mice at 3 weeks after surgery.At 2 weeks after tumor inoculation, mice were euthanized, and peritoneal metastases were assessed based on macroscopic nodules on the mesentery.All procedures were approved by the Animal Care Committee of Jichi Medical University (approval no.21057-01) and performed in accordance with the ARRIVE guidelines and the Japanese Guidelines for Animal Research.

Flow cytometry
For flow cytometry analysis, whole blood was harvested, red blood cells were eliminated with lysis buffer (NH 4 Cl, KHCO 3 , EDTA 4Na), and single-cell suspensions were prepared.Suspensions were adjusted to 1 × 10 6 cells per 100 µL in PBS containing 0.02% EDTA, and incubated for 15 min at room temperature to label dead cells using FVS780.Cells were washed with PBS, incubated with 5 µL FcR blocking reagent for 10 min at 4 °C, and immunostained with relevant mAbs according to the manufacturer's instructions.Cells were washed with PBS and then underwent flow cytometry (LSRFortessa X-20, Becton Dickinson, San Jose, CA, USA) for analysis of antigen expression by using Flow Jo software (Becton Dickinson).The absolute cell counts in 1 ml of blood were calculated by multiplying the specific subset frequency by the live cell counts obtained from microscopic observation using trypan blue staining.

Sequential immunohistochemistry of subcutaneous primary tumors and lung samples
Mice were euthanized at day 28 after inoculation with tumor cells; subcutaneous primary tumors and lungs were excised and fixed with 4% formalin.Lungs also underwent internal fixation.Paraffin-embedded 4-µm representative central sections of samples were prepared for immunohistochemical evaluation and staining with hematoxylin and eosin.
Sequential immunohistochemistry was performed as previously described 60 .Briefly, after deparaffinization in xylene and rehydration through a graded series of ethanol immersion baths, sections were washed with running water for 5 min.After rehydration, slides were stained with hematoxylin for 1 min and then coverslips were applied with VectaMount AQ Aqueous Mounting Medium (Vector Laboratories, Newark, CA, USA).Slide then underwent whole-tissue scanning (OlyVia Slideview VS200, Olympus, Tokyo, Japan), after which coverslips were removed by immersion in Tris-buffered saline with 0.1% Tween 20 (TBST).
After scanning, the coverslips were removed by using TBST and the slides were treated with a gradient of 70%, 80%, and 90% ethanol for every 2 min for destaining.Slides were incubated until no visible AEC reaction product remained.After destaining, antibodies were eluted by incubating sections in Instant Buffer Phosphate Buffer Solution (As One) for 10 min using the microwave method.The tissues were then revitalized, starting again with the blocking step, as described earlier.Complete stripping of antibodies and signals throughout all cycles was confirmed.
For evaluating the densities of these immune cells, we used a region of interest (ROI) of 1.0 × 1.0 mm for primary tumors and lung tissue; for lung metastatic nodules, we used an ROI of 0.3 × 0.3 mm for metastatic lesions larger than 160,000 µm 2 (totals: 85 lesions in splenectomized mice, 26 in the sham group).Two investigators blinded to treatment group independently used ImageJ Fiji to count these cells in 3 random microscopic fields; they then calculated the density of each cell type (number/mm 2 ).

Statistical analysis
Statistical analysis was performed by using Prism 8 (Graph Pad Softeare, San Diego, CA, USA).Data are shown as median values or mean ± 1 standard deviation (SD) and were compared by using the Mann-Whitney U test.Correlation was evaluated through Pearson simple linear regression analysis.In all analyses, P < 0.05 was considered statistically significant.

Animal study
The protocol of animal study was approved by Institution Review Board of the Animal Care Committee of Jichi Medical University (Approval No. 21057-01).

Figure 1 .
Figure 1.Experimental scheme and primary tumor growth.(A) BALB/C mice underwent splenectomy or a sham operation prior to subcutaneous injection of LuM1 colon adenocarcinoma cells (1 × 10 6 per mouse) in the right flank.(B) Body weight and (C) tumor volume were measured twice each week after tumor inoculation.On day 28, mice were euthanized, and (D) primary tumors were weighed.The combined results of 2 experiments (n = 7 or 8 mice in each group) are shown.*P < 0.05 (Mann-Whitney U test).

Figure 2 .
Figure 2. Growth of macroscopic and microscopic metastases in lung.(A, B) On day 28, lung metastases were evaluated by counting the number of macroscopic nodules visible at the lung surface.The combined results of 2 experiments (n = 7 or 8 mice in each group) are shown.(C, D) Representative coronal tissue sections from the central part of formalin-fixed lung specimens were stained with hematoxylin and eosin, and the total number of lung metastases, including microscopic lesions, was counted.**P < 0.01 (Mann-Whitney U test).

Figure 5 .
Figure 5. Macroscopic lung metastases and circulating immune cells.Correlation between the number of macroscopic lung metastases and the percentage of (A) CD49b + CD335 + NK cells or (B) CD11b + Gr-1 - Ly6c -CX3CR1 + patrolling monocytes in blood was evaluated by using Pearson's correlation coefficient.(C) The correlation between the percentages of NK cells and patrolling monocytes was evaluated also.Pearson simple linear regression analysis was used to calculate r and P values.
, was cultured in DMEM supplemented with 10% et al bovine serum, 50 U/mL penicillin, and 50 µg/mL streptomycin.Once they had achieved > 80% confluence, cells were detached through treatment with TrypLE Express (Thermo Fisher Scientific, Waltham, MA, USA).Cultured cells were tested every 3 months by using the Mycoplasma Detection Kit (R&D Systems, Minneapolis, MN, USA) and used for experiments after three passages.